The present invention relates to peptides (proteins) having cell-proliferative, cell-differentiating and/or cell-protective properties referred to as cadherin-derived growth factor (CDGF), and their use.
It has been the object of the invention to provide peptides having cell-proliferative, cell-protective and/or cell-differentiating properties.
This object is achieved by peptides referred to as cadherin-derived growth factor (CDGF) the sequence of which corresponds to a partial sequence of a pre-pro-cadherin, said pre-pro-cadherin comprising the domains signal sequence, pro sequence, cadherin repeats, transmembrane region and intracellular domain, characterized in that the sequence of said peptide comprises the pro sequence, that at least one of the other domains of the pre-pro-cadherin is lacking, and that said peptide has cell-proliferative, cell-protective and/or cell-differentiating properties.
For example, the cell-proliferative activity can be determined on primary osteoblasts from rat calvarias, and the cell-protective and/or cell-differentiating activities can be determined on primary nerve cell cultures from spinal ganglia of chicken embryos.
In a preferred embodiment, the CDGF is a fragment of pro-cadherin, i.e., of pre-pro-cadherin truncated by the signal sequence.
More preferably, it is the N terminus of pro-cadherin, i.e., the part cleaved off during the processing of pro-cadherin into cadherin, or an N-terminally or C-terminally truncated fragment thereof.
Preferably, the CDGF comprises no cadherin repeats.
Preferred embodiments according to the invention are peptides having the sequence:
Cadherin-1 human (28-154) (SEQ ID NO: 1):
CHPGFDAESYTFTVPRRHLERGRVLGRVNFCTGRQRTAYFSLDTRFKVGTDGVITVKR-PLRFHNPQIHFLVYAWDSTYRKFSTKVTLNGHHHRPPPHQASVSGIQAELLTFPNSSP-GLRRQKR
Cadherin-2 human (24-15) (SEQ ID NO: 2):
EASGEIALCKTGFPEDVYSAVLSKDVHEGQPLLNVFSNCNGKRKVQYESSEPADFKVD-EDGMVYAVRSEPLSSEHAKFLIYAQDKETQEKWQKLSLKPTLTEESVKESAEVEEIVF-PRQFSKHSGHLQRQKR
CadIicrin-3 human (27-107) (SEQ ID NO: 3):
CP.AV FREAFEVTLEAGGAEQEPGQALGKVFMGQEPALFSTDNDDFTVRNGETVQER-RSLKERNPLKIFPSKRILRRHKR
Cadherin-4 human (21-169) (SEQ ID NO: 4):
HNEDLTTRETCKAGFSEDDYTALISQNILEGEKLLQVKSSCVGTKGTQYETNSMDFKG-ADGTVFATRELQVPSEQVAFTVTAWDSQTAEKWDAVLVAQTSSPHSGHKPQKGKKVVALDPSPPPKDTLLPWPQHQNANG
Cadherin-5 human (26-47) (SEQ ID NO: 5):
AGANPAQRDTHSLLPTHRRQKR
Cadherin-6 human (19-53) (SEQ ID NO: 6):
TLSTPLSKRTSGEPAKKRALELSGNSKNELNRSKR
Cadherin-6 human (1xe2x80x29-51) (SEQ ID NO: 7):
TLSTPLSKRTSGFPAKKRALELSGNSKNELNRS
Cadherin-S human (SEQ ID NO: 8):
MLLDLWTPLIILWITLPPCIYMAPMNQSQVLMSGSPLELNSLGEEQRILNRSKR
Cadherin-B human (Cadherin-11) Precursor (23-53) (SEQ ID NO: 9):
FAPERRGHLRPSFHGHHFKGKEGQVLQRSKR
Cadherin-B human (Cadherin-11) Precursor (26-51) (SEQ ID NO: 10):
ERRGHLRPSFHGHHEKGKEGQVLQRS (OB-CDGF)
Cadherin-C human (Cadherin-12)-Brain-Cadherin Precuirsor (24-54) (SEQ ID NO: 11):
QPQPQQTLATEPRENVIHLPGQRSHFQRVKR
Cadherin-C human (Cadherin-12)-Brain-Cadherin Precursor (24-52) (SEQ ID NO: 12):
QPQPQQTLATEPRENVIHLPGQRSHFQRV
Cadherin-D human (Cadherin 13) (23-138) (SEQ ID NO: 13):
EDLDCTPGFQQKVFHINQPAEFIEDQSILNLTFSDCKGNDKLRYEVSSPYFKVNSDGG-LVALRNITAVGKTLFVHARTPHAEFDMAELVIVGGKDISLQDIFKFARTSPVPRQKRP-SVLLLSLFSLACL
Cadherin-F human (Cadherin 14) (22-60) (SEQ ID NO: 14):
VPGWRRPTTLYPWRRAPALSRVRRAWVIPPISVSENHKR.
Preferably, the protein according to the invention is a protein obtainable from humans or a naturally occurring human variant thereof.
The present invention also relates to a novel protein which comprises parts of the amino acid sequence of CDGF. The invention preferably relates to a CDGF which contains the above represented amino acid sequences, but may also contain variants of these sequences. The term xe2x80x9cvariantsxe2x80x9d as used herein relates to sequences which are distinguished from the above represented amino acid sequences of CDGFs by the substitution, deletion and/or insertion of individual amino acids or short amino acid sequences.
The term xe2x80x9cvariantsxe2x80x9d encompasses both naturally occurring allelic variations of the CDGFs and proteins generated by recombinant DNA technology (especially by in vitro mutagenesis using chemically synthesized oligonu-cleotides) whose biological and/or immunological activities correspond to those of the CDGFs.
Conservative exchanges include, for example, Y for V or vice versa, K for S or vice versa, A for S or vice versa, D for E or vice versa, G for S or vice versa, R for Q or vice versa, R for A or vice versa, Q for K or vice versa.
According to the invention, there are also claimed nucleic acids coding for said peptides or derivatives, or those complementary to such nucleic acids. The nucleic acids may be, for example, DNA, RNA, PNA or nuclease-resistant analogues thereof. The nucleic acids according to the Invention are suitable for the in vivo expression of the CDGFs in a gene therapy, and as antisense nucleotides for reducing expression. The invention also relates to vectors containing said nucleic acids. The vectors are suitable, in particular, for expressing the peptide in genetically engineered organisms.
Further, the invention relates to antibodies directed against CDGF or derivatives thereof, and antagonists/inhibitors directed against CDGF, a derivative thereof, or any of the nucleic acids according to the invention. These substances are useful as medicaments for treating conditions related to an overexpression of CDGF, and for use in diagnostics.
The CDGFs, derivatives, compounds, nucleic acids, antibodies and/or antagonists/inhibitors according to the invention can be used as medicaments together with usual auxiliary agents. It is particularly preferred for the medicaments to be incorporated in suitable galenic formulations for oral, buccal, intravenous, intramuscular, intracutaneous, intrathecal, intranasal, topical administrations, and as an aerosol for transpulmonary administration.
The amount of medicament to be administered is preferably from 1 xcexcg to 1 g per dosage unit per day.
The medicaments according to the invention are suitable for the treatment and prophylaxis of degenerative and metabolic diseases of the bones, such as osteoporosis, osteomalacia and osteopenia, of the pancreas, such as diabetes mellitus, of the muscles, such as muscular dystrophies, of the vessels, of the central and peripheral nervous systems, such as peripheral and central neuropathies, of the lungs, such as bronchial asthma, of the stomach, such as ulcer, and for the therapy and prophylaxis of inflammatory processes, disturbed inflammatory reactions, tumor diseases, and for wound and bone healing.
The diagnostic agent according to the invention contains poly- or mono-clonal antibodies against the peptide according to the invention, optionally in a labeled form, such as fluorescence-labeled or radioactively labeled forms, to be used in a per se known ELISA or RIA. The diagnostic agent according to the invention contains DNA, RNA and/or PNA, optionally in a modified and/or labeled form, for use in test systems known to those skilled in the art, such as PCR or fingerprinting.
The diagnostic agents are suitable for checking CDGF levels in tissues, in secretions and/or in body fluids, such as plasma, urine and cerebrospinal fluid, and as markers for functional disorders in bones, muscles, vessels, the nervous system, lymph organs, the gastrointestinal tract, the immune system, and of diabetes and inflammatory and neoplastic processes, and as a marker in cancer (tumor marker).
The CDGFs according to the invention and their derivatives can be obtained by isolation from hemofiltrate using cation-exchange extraction followed by elution of the adsorbed substances, renewed cation-exchange chromatography of the extract containing the peptides, and multistage reversed-phase chromatography. In total synthesis, the peptides according to the invention can be obtained by solid-phase synthesis in terms of Merrifield synthesis, or liquid-phase synthesis according to methods involving protected amino acids, known to those skilled in the art, followed by purification. The peptides according to the invention can also be prepared by methods of heterologous expression, known to those skilled in the art, using common biotechnological vectors.
For example, a peptide referred to as OB-CDGF was purified from human hemofiltrate by chromatographical methods and identified using a bioassay.
The peptide has a molecular mass of 3062.8 Da. To date, from the analysis of a cDNA, an OB-cadherin pre-pro sequence has been postulated. In this sequence derived from the cDNA, the peptide sequence according to the invention is located directly behind the putative signal sequence (see FIG. 1).
The biochemical characterization of the peptide according to the invention was effected by mass spectrometry and sequencing of the whole peptide. The sequence analysis of the biologically active peptide yielded the following-amino acid sequence for OB-CDGF:
ERRGHLRPSFHGHHEKGKEGQVLQRS (SEQ ID NO: 10)
In the ESI (electrospray ionization) mass spectrum of the OB-CDGF, its molecular weight was determined to be:
OB-CDGF, MW 3062.8 Da.
The peptide according to the invention can be obtained by a purification method starting from human hemofiltrate. This method according to DE 36 33 707, which discloses the recovery of proteins from hemofiltrate, was performed in a modified form.
Hemofiltrate is obtained in large amounts in the ultrafiltration of the blood of kidney disease sufferers. The human hemofiltrate is optionally diluted with water and acidified. Its pH value is preferably from 1.5 to 3.5, especially from 2.5 to 3.0. Thereafter, the hemofiltrate is passed over a cation exchanger, for example, a support material modified with sulfonic acid groups (Fraktogel SP-650 (M), Merck, Darmstadt). The peptides bound to the cation exchanger are eluted with a relatively highly concentrated salt solution. The ionic strength of the eluent is about that of a 0.5 to 1 M ammonium acetate solution.
The collected eluate is subjected to another cation exchange chromatography. This chromatography is preferably a stepwise elution with buffers of increasing pH values.
The fractions containing the peptide according to the invention are further purified by preparative reversed-phase chromatography followed by semi-preparative reversed-phase chromatography, for example, on C4-modified support materials. The purification level is preferably determined by analytical reversed-phase chromatography on C18-modified support materials.
The substance obtained by the chromatographic purification was subjected to structural elucidation. The molecular weight of the native peptide was determined using an electrospray mass spectrometer. The sequence analysis was effected through Edman degradation of the peptides and of chemically modified derivatives with an ABI 473 A sequencer.
Total synthesis was effected on usual solid phases according to Merrifield synthesis. The synthetic strategy and the construction of the peptide and derivatives thereof using the correspondingly protected amino acids are known to those skilled in the art.
The OB-CDGF and its cDNA, its gene and analogues, fragments and derivatives of said peptide, cDNA and gene as well as antibodies antagonizing the OB-CDGF can be used as medicaments.